Target product profile: Trypanosoma brucei gambiense test for low-prevalence settings

Abstract Having caused devastating epidemics during the 20th century, the incidence of life-threatening human African trypanosomiasis has fallen to historically low levels as a result of sustained and coordinated efforts over the past 20 years. Humans are the main reservoir of one of the two pathogenic trypanosome subspecies, Trypanosoma brucei gambiense, found in western and central Africa. The expected advent of a safe and easy-to-use treatment to be given to seropositive but microscopically unconfirmed individuals would lead to further depletion; in the meantime, the presence of T. b. gambiense infection in the community must be monitored to allow the control strategy to be adapted and the elimination status to be assessed. The World Health Organization has therefore developed a target product profile that describes the optimal and minimal characteristics of an individual laboratory-based test to assess T. b. gambiense infection in low-prevalence settings. Development of the target product profile involved the formation of a Neglected Tropical Diseases Diagnostics Technical Advisory Group and a subgroup on human African trypanosomiasis diagnostic innovation needs, and an analysis of the available products and development pipeline. According to the product profile, the test should ideally: (i) require a minimally invasive or non-invasive specimen, collectable at peripheral facilities by minimally trained health workers; (ii) demonstrate good sensitivity and high specificity; (iii) have a stability of samples allowing transfer to reference laboratories preferably without cold chain; (iv) be stable over a wide range of environmental conditions for more than 2 years; and (v) after marketing, be available at low cost for at least 7 years.


Introduction
Human African trypanosomiasis, endemic in sub-Saharan Africa, is a life-threatening parasitic infection transmitted by the tsetse fly. 1 Having caused devastating epidemics during the 20th century, its incidence has fallen to historically low levels as a result of sustained and coordinated efforts over the past 20 years. 2 The disease is caused by two trypanosome subspecies with distinct epidemiology. (i) Trypanosoma brucei rhodesiense, found in eastern and southern Africa, is harboured by wild and domestic animals (which constitute its reservoir) and is occasionally transmitted to humans; and (ii) T. b. gambiense, which is found in western and central Africa, has its main reservoir in humans and between 2011 and 2020 accounted for 95% (32 275 out of 34 096 reported cases) of the total caseload of human African trypanosomiasis. 2 Because clinical signs and symptoms of human African trypanosomiasis are unspecific, diagnosis relies on laboratory techniques. 3 Field serodiagnostic tests exist only for T. b. gambiense and are based on the detection of antibodies; they are therefore not confirmatory of infection. With the current low disease prevalence, the positive predictive value of serological tests is particularly low. Field-applicable tools include the card agglutination test for trypanosomiasis, 4 used mainly in active screening by specialized mobile teams, and the rapid diagnostic tests that are more suitable for individual testing at point-of-care. Confirmation of T. b. gambiense infection requires microscopic examination of body fluids, necessitating specific training. The best performing methods are laborious and demonstrate 85%−95% diagnostic sensitivity when performed by skilled personnel. 5 With regards to the control and elimination of gambiense human African trypanosomiasis, it has long been observed that a strategy of repeated rounds of screening followed by treatment of detected cases can bring down the prevalence substantially. 6 The expected advent of a safe and easy-to-use treatment to be given to seropositive but microscopically unconfirmed individuals would lead to further depletion of the parasite reservoir. 7 However, if this type of treatment policy is applied, the presence of T. b. gambiense infection in the community must be monitored to allow the control strategy to be adapted in each setting and to provide key data to assess the elimination status in endemic countries.
The World Health Organization (WHO) has therefore developed a target product profile that describes the optimal and minimal characteristics of an individual laboratory-based test to assess T. b. gambiense infection in low-prevalence settings. The purpose of this profile is to inform product developers of the key performance specifications and characteristics of such a test that will meet the needs of end-users in sub-Saharan Gambiense human African trypanosomiasis individual test Gerardo Priotto et al.
African countries. The main features of such a product include: (i) its use at an individual level in suspects (e.g. those with serological or clinical suspicion, and/or geographical proximity to confirmed cases) to determine if they are currently infected (ideally) or have previously been infected by T. b. gambiense; (ii) high specificity and good sensitivity; (iii) simple specimen collection by minimally trained staff, and stability of samples allowing transfer to reference laboratories preferably without cold chain; and (iv) ideally requiring blood from finger-prick with minimal sample processing. To help with interpretation, it should also be established for how long the test may remain positive in an individual after a T. b. gambiense infection has cleared (e.g. antibody tests may remain positive for years).

Methods
The development of this target product profile was led by the WHO Department of Control of Neglected Tropical Diseases following standard WHO guidance.
To identify and prioritize diagnostic needs, a WHO Neglected Tropical Diseases Diagnostics Technical Advisory Group was formed. Subgroups to advise on specific neglected tropical diseases were subsequently created, including a subgroup working on human African trypanosomiasis diagnostic innovation needs. This group of independent experts comprised leading international scientists and specialists, including from countries where the disease is endemic, who followed standard WHO declaration of interest procedures. A landscape analysis of the available products and development pipeline was conducted, and salient areas with unmet needs were identified. Through meetings and remote consultations, use-cases were developed for the hypothetical tools considered as the four main gaps, and assigned an order of priority. A template adapted to the human African trypanosomiasis context was agreed, and used to develop the target product profile. The draft of this target product profile (rated as third-highest priority) underwent several rounds of review by the subgroup members, before being reviewed by the members of the advisory group. A draft version was posted on the WHO website for public consultation on 23 September 2022 for 28 days, inviting viewers to complete a proforma comment form. After further review by members of the advisory group, a final version of the target product profile was made available online. 8

Target product profile Intended use
This test is intended for individuals at increased suspicion for gambiense human African trypanosomiasis after serological testing or clinical examination, or because of proximity to a confirmed case. Ideally, a positive test should indicate current infection with gambiense human African trypanosomiasis, with inclusion of a previous infection as acceptable.
It would be desirable for a test to detect the presence of T. b. gambiensespecific antibodies, T. b. gambiense antigens, or whole-parasite or T. b. gambiense-specific nucleic acids; however, the detection of antibodies or antigens of Trypanozoon, or whole-parasite or Trypanozoon-specific nucleic acids, would be minimally acceptable. The sampling should be conducted before treatment in the case of molecular or antigen analytes. Antibodies may persist from a previous infection, which would allow the retrospective diagnosis of T. b. gambiense infection; sampling could be conducted immediately after treatment in this case.
The type of specimen required should ideally be either minimally invasive (e.g. finger-prick or venous blood) or non-invasive (e.g. saliva, urine or tears). A test feasible at a national or sub-national laboratory would be ideal, but the need to refer to international laboratories would be minimally acceptable. A trained health worker (with ideally < 1 hour's basic training, or acceptably up to 4 hours) should be capable of collecting the specimens. At the reference laboratory performing the test, a 1-2 days' training for laboratory personnel should be sufficient, and a maximum of 7 days would be acceptable.

Assay performance
A clinical sensitivity of at least 95% is desired (> 90% minimally acceptable), comparable to the most sensitive parasitological tests currently in use. False negatives resulting in non-treatment not only have a risk of death but also result in lower prevalence estimates. Further, because false positives result in unnecessary treatment and overestimated prevalence, a clinical specificity of at least 99% is desired (> 95% minimally acceptable).
Because diagnosis and treatment are currently based on microscopy at the subgenus level, a Trypanozoon-specific test would be acceptable; however, the test should ideally detect lower taxa (e.g. T. b. gambiense type 1). In terms of detection thresholds, an analytical sensitivity of 10 parasites/mL or less is desired (although ≤ 50 parasites/mL would be minimally acceptable). Repeatability and reproducibility should be as high as possible, with Κ-values of greater than 0.96 and 0.94 desired, respectively (> 0.92 and > 0.90 minimally acceptable, respectively). Control of functionality is required, and the availability of temperature-stable positive and negative controls for batch and kit testing is desired.

Operational characteristics
The test should ideally be usable in conditions of 10 °C-40 °C and 10%-88% relative humidity, although margins of 10°C -30 °C and 40%-70% would be minimally acceptable. Preparation of the specimen in the field should preferably be a single-step process (maximum four steps), with preferably no (or minimal) need for precision liquid handling or specialized material. Specimens should ideally be testable individually (i.e. without any waste of unused reagent), although testing in batches of less than eight would be minimally acceptable. Results should ideally be available within 48 hours (maximum 1 week) after arrival at the test laboratory, preferably with automatic scoring, recording and integration of results (visual/manual scoring, recording and integration minimally acceptable).

Reagent and sample handling
The stability of the reagent should consider the time frame for distribution from manufacturer, passage through customs and the limited number of tests that may need to be performed in low-prevalence settings. Tests should be stable at 4 °C-45 °C (4 °C-8 °C minimally acceptable) and 40%-88% relative humidity, ideally for more than 2 years (1 year minimally acceptable). Ideally, individual tests (or < 8-specimen series) should be accompanied by all necessary accessories and reagents for processing.

Gambiense human African trypanosomiasis individual test Gerardo Priotto et al.
Reagents should either be ready for use, or else usable after a preferred maximum of two steps (maximum five steps minimally acceptable).
Specimen processing (volume ≤ 0.07 mL for finger-prick; ≤ 5 mL for venous blood) before preferably nonurgent transport (e.g. 4 weeks) should ideally be achievable either without any further steps or a maximum of two steps. Stability of the sample for 4 weeks at 40 °C or 12 months at 4 °C is desirable; stability of the sample for 3 days at 35 °C or 1-2 months at 4 °C would be minimally acceptable. Sample waste management procedures should adhere to standard biosafety measures for handling potentially infectious materials, with waste disposal in biosafety bins and sharps containers following standard guidelines.

Commercial aspects
The supply of the test should be guaranteed for preferably 7 years or more (at least 5 years minimally acceptable) after marketing, and manufacturers should replace non-functioning tests or instruments. External support should be available with a desired response time of ideally 1 day (1 week maximum).
Individual tests should be available to health facilities at a maximum cost of 5 United States dollars (US$); the minimally acceptable cost is under US$ 20.

Conclusion
Currently, the diagnostic tools available in or near the field are not appropriate for determining T. b. gambiense infection because of the low sensitivity of simple microscopy tests, and the low feasibility of more sophisticated tests with higher performance. According to the product profile, the test should ideally: (i) require a minimally invasive or non-invasive specimen, collectable by fixed or mobile facilities close to affected communities by minimally trained health workers; (ii) demonstrate good sensitivity, high specificity, repeatability and reproducibility; (iii) have a simple specimen collection procedure, and a good stability of specimens allowing transfer to reference laboratories preferably without cold chain ; (iv) be stable over a wide range of temperature and relative humidity for more than 2 years; and (v) after marketing, be available at low cost for at least 7 years. In settings with an absence of performant microscopy, treatment could be decided based on the results of this test. This test would also become very useful in the framework of a potential strategy of providing immediate treatment to unconfirmed serological suspects, a situation that is currently predicted with the potential advent of an oral, single-dose safe treatment. In such situations, this test could confirm or rule out T. b. gambiense infection a posteriori, allowing the epidemiological situation to be monitored. ■